U.S. patent number 4,994,446 [Application Number 07/293,136] was granted by the patent office on 1991-02-19 for drug system.
This patent grant is currently assigned to Ramot - University Authority for Applied Research and Industrial. Invention is credited to Yoel Kloog, Mordechai Sokolovsky.
United States Patent |
4,994,446 |
Sokolovsky , et al. |
February 19, 1991 |
Drug system
Abstract
The invention relates to a drug system for the treatment and
alleviation of the symptoms of epilepsy; as anticonvulsant agent,
and for the prevention or alleviation of brain damage due to
strokes. The drug system comprises as active ingredient a compound
such as MK-801, phencyclidine or as thienyl-cyclohexyl-piperidine
(TCP), which is administered in such a manner that it encounters
and binds to the N-methyl-D-aspartate (NMDA) receptor channels.
This is in the presence of glutamate or glycine, or similarly
acting amino-acid, which is either present in the patient, or which
can be administered separately. The effective drug can be
effectively locked in the receptor channels by means of a glutamate
antagonist, such as AP-5.
Inventors: |
Sokolovsky; Mordechai (Tel
Aviv, IL), Kloog; Yoel (Herzelia, IL) |
Assignee: |
Ramot - University Authority for
Applied Research and Industrial (Tel Aviv, IL)
|
Family
ID: |
23127801 |
Appl.
No.: |
07/293,136 |
Filed: |
January 3, 1989 |
Current U.S.
Class: |
514/75;
514/326 |
Current CPC
Class: |
A61K
31/66 (20130101); A61K 31/66 (20130101); A61K
31/445 (20130101); A61K 31/495 (20130101); A61K
31/66 (20130101); A61K 2300/00 (20130101) |
Current International
Class: |
A61K
31/66 (20060101); A61K 031/66 (); A61K
031/441 () |
Field of
Search: |
;514/75,326 |
Other References
Chem. Abst. 108 126054n, (1988). .
Chem. Abst. 110 128473p, (1989)..
|
Primary Examiner: Friedman; Stanley J.
Attorney, Agent or Firm: Browdy & Neimark
Claims
We claim:
1. A pharmaceutical composition for the treatment of epilepsy, for
use as anticonvulsant and for the prevention of damage caused by
strokes in mammals, including humans, which comprises in
combination from 15 mg to 500 mg phencyclidine and from 350 mg to
1000 mg of D (-) 2-amino-5-phosphovaleric acid (AP-5).
2. A method for the treatment of epilepsy, for treating convulsions
and for preventing the damage to the brain caused by a stroke, or
at least for substantially reducing such damage, which comprises
administering to the patient in need thereof a pharmacologically
effective quantity of a pharmaceutical composition claimed in claim
1.
3. A method according to claim 2, where for the removal of the
effective drug from the channel, there is administered an excess of
glutamate.
Description
FIELD OF THE INVENTION
The present invention relates to a novel drug system for use in the
treatment of, and for the alleviation of the symptoms of a variety
of diseases and states of ill-health in mammals, and especially in
humans. Amongst others the drug system is of use an antiepileptic,
as anticonvulsant, for the preparation or alleviation of brain
damage caused by strokes etc.
The drug system is based on a combination of drugs of the MK-801
and PCP type, in combination with, or in sequential administration
of certain excitatory amino acids, such as glutamate, glycine and
aspartate, as well as related analogs, which substantially increase
the rate of binding of the effective drug to the specific receptor.
MK-801 is bound to the N-methyl-D-aspartate receptor, and it would
seem that MK-801 acts as steric blocker of the NMDA channel. When a
prolonged action of the drug is required, there is further
administered an antagonist to such amino-acid, such as a glutamate
antagonist, resulting in the freezing of the drug in the
channel.
BACKGROUND OF THE INVENTION
The activity of PCP (phencyclidine) is discussed in NIDA Notes 2
(1987) 9. This article sets out the binding of PCP to two different
receptors of the nerve membranes. MK-801,
(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine
maleate is known as potent anticonvulsant, and it is also known
that MK-801 is a potent N-methyl-D-aspartate (NMDA) antagonist:
Proc. Nat. Acad. Sci. U.S.A. 83 (1986) 7104. The kinetic
characterization of the phencyclidine-NMDA receptor interaction,
setting out evidence of a steric blockage of the channel was
described in Biochem. 27 (1988) 843. It is known that there exist a
number of excitatory amino acid antagonists, some of which block
the neurotoxic activity of N-methyl-aspartate (NMDA). Amongst the
most effective of these are phencyclidine (PCP) and MK-801, see
Europ. J. Pharmac. 141 (1987) 357.
SUMMARY OF THE INVENTION
The present invention relates to a novel drug system which can be
used in a variety of illnesses. The drug system is valuable as
antiepileptic, anticonvulsant and agents that will prevent brain
damage caused by strokes.
The novel system is based on a combination of an effective drug of
the PCP and MK-801 and TCP (which is
N[1-(2-thienyl)cyclohexyl]-piperidine, with a certain aminoacid or
mixture of amino acids of the excitatory aminoacid type, such as
glutamate, glycine and asparate, and means for fixing the effective
drug in a receptor channel, and means for the removal of the drug,
if required, from such channel.
The active drugs (PCP, TCP, MK-801) seem to be steric blockers of
the receptor channels, and especially of the NMDA channels. When
administered simultaneously, or in a close sequence with glutamate,
glycine or amino acids with the same type of activity, result in a
considerable enhancement of the rate of binding of the
drug-receptor complex. When it is desired to prolong the action of
the drug, there is administered a suitable receptor antagonist,
such as AP-5 which is a NMDA-receptor antagonist. This seems to
inactivate the amino acid receptor, freezing the drug in the
channel. The ratio of glutamate to TCP, for example, is of the
order of .mu.-molar of glutamate to 100 n-molar for TCP or MK-801.
When the drug is already affixed in the receptor channels, and it
is desired to terminate its activity, by its removal, there is
administered an excess of glutamate, which again opens the channel,
facilitating the exit of the drug, which is rapidly removed from
said channels. Thus, when MK-801 is administered as antiepileptic
drug or as agent to prevent stroke damage, it is administered with
an excess of an amino acid such as glutamate, and after its entry
into the receptor channel, it is blocked therein by the
administration of an antagonist to such amino acid, such as the
glutamate antagonist D-(-)2-amino-5-phospho-valeric acid, AP-5.
Similar results are obtained by the administration of PCP and TCP,
in the presence of suitable amino acids adapted to open up the
receptor channels. Kinetic and equilibrium binding experiments
demonstrate that TCP and MK-801, as well as PCP are steric blockers
of the channel.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings illustrate the invention. In such
drawings:
FIG. 1 is a graph that illustrate the time course of association of
MK-801 with the NMDA receptor channel.
FIG. 1A is a graph that illustrates the increase in ligand-receptor
binding as being time dependent.
FIG. 1B is a graph that illustrates the observed time constants
vary in a linear fashion with [.sup.3 H]MK-801 concentrations.
FIG. 2 is a graph that illustrates the equilibrium binding of
MK-801.
FIG. 2A is a graph that illustrates first order plots of the
dissociation of MK-801 receptor complexes.
Abbreviations
phencyclidine (PCP); N[1-(2-thienyl)cyclohexyl]piperidine (TCP);
N-methyl-D-aspartate (NMDA); D(-)2-amino-5-phosphovaleric acid
(AP-5); (+) 5 methyl-10,11
dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate
(dibenzocycloalkenimine) (MK-801).
Binding assays were performed in triplicate at 25.degree. C. with
repeatedly washed rat cerebral cortex membranes (80 .mu.g protein)
as described in Biochem. ibid. Assays were carried out in the
absence or in the presence of 10.sup.-4 M AP-5, or in the presence
of 10 .mu.M MgCl.sub.2 together with 1 .mu.M L-glutamate and 1
.mu.M glycine. Nonspecific binding was determined in samples
containing 10.sup.-4 M PCP. Data were expressed as the specific
binding of [.sup.3 H]MK-801 and analyzed according to the two-step
binding model of interactions between noncompetitive blockers and
the NMDA receptor channel. The model assumes a first order
diffusion of the ligand from the outside into the interior of the
channel, followed by association of the ligand with its receptor
sites. Under the two extreme conditions employed here, viz.,
binding of [.sup.3 H]MK-801 in the presence of agonists (unlimited
diffusion) and in the absence of agonists (limited diffusion) the
time courses of receptor occupation should respectively follow
pseudo first order (eq. 1) and first order (eq. 2) kinetics:
where L, R.sub.T, RLeq and RL are respectively the ligand, the
total number of binding sites, the bound ligand at equilibrium and
at time t. k.sub.a and k.sub.b are the forward and background
diffusion constants; k1 and k.sub.- 1 are the second order on-rate
and the first order off-rate constants for the binding process. The
overall equilibrium binding constant (K.sub.d) is given by K.sub.-
1.multidot.k.sub.b /k1.multidot.k2.
FIG. 1A demonstrates typical time courses for [.sup.3
H]MK-801-receptor association, with and without glutamate and
glycine. The association rate of 5 nM [.sup.3 H]MK-801 with the
receptor in the absence of exogenous agonists was very slow
(t1/2.perspectiveto.70 min) and reached equilibrium only after a
prolonged incubation time (>6 hr). Upon addition of glutamate
and glycine the rate of [.sup.3 H]MK-801 binding to the receptor
was markedly increased (t1/2.perspectiveto.7 min) and the reaction
approached equilibrium within 45-60 min. The increase in
ligand-receptor binding was time dependent; immediately after the
onset of binding it was very high (.about.9 times that of the
control) and then it declined (FIG. 1A). Similar time courses (not
shown) were followed by 2 nM and 8.6 nM [.sup.3 H]MK-801. The
kinetics of [.sup.3 H]MK-801 binding in the presence of glutamate
and glycine followed a pseudo first order scheme (eq. 1): the
observed time constants (K.sub.obs ) varied linearly with [.sup.3
H]MK-801 concentrations (FIG. 1B), and the ratio between the
apparent on-rate time constant (1.55.multidot.10.sup.7 M.sup.-1
min.sup.-1) and the apparent off-rate time constant (0.075
min.sup.-1) was 4.8 nM. This kinetically derived dissociation
constant (K.sub.d) was similar to the K.sub.d for [.sup.3 H]MK-801
determined at equilibrium in the presence (4.2 nM) and in the
absence (2.1 nM) of glutamate and glycine (FIG. 2).
In the absence of exogenous agonists the kinetics of
ligand-receptor association did not follow a pseudo first order
scheme, as shown by the fact that the half times of the reactions
(60-70 min) did not vary as a function of [.sup.3 H]MK-801
concentration. These kinetics fitted well to the first order
reaction scheme (eq. 2), and the observed time constant (k.sub.b)
derived from the kinetics of ligand receptor association
(2.28.multidot.10.sup.-3 min.sup.-1, FIG. 1C) was similar to the
dissociation time constant obtained when receptor-ligand
dissociation was measured in the absence of agonists
(5.5.multidot.10.sup.-3 min.sup.-1, FIG. 2). Also, the addition of
1 .mu.M glutamate to [.sup.3 H] 801-receptor complexes resulted in
a marked increase in the dissociation rate (half time 18 min,
compared to the control value of 180 min, FIG. 2) and the effect
was enhanced by glycine (half time=4 min, FIG. 2). Consistency with
the two-step binding model (see Methods) was further shown by the
fact that the dissociation rate constant determined in the presence
of glutamate and glycine (0.1 min.sup.-1, FIG. 2) was similar to
k.sub.-1 value obtained under the same conditions during
ligand-receptor association (0.075 min.sup.-1, FIG. 1B). Taken
together, the association and dissociation kinetics of [.sup.3 H]
801 binding to the receptor suggest that in the absence of agonists
the rate-limiting step is the diffusion of the outside ligand
(L.sub.o) into the interior (L.sub.c) of the presumably closed NMDA
receptor channel ##STR1## whereas in the presence of agonists this
limitation is removed (presumably because the channel is open) and
the rate-limiting step is the binding process itself. ##STR2##
It should be noted, however, that the competitive NMDA-receptor
antagonist, AP-5, by itself decreases the rates of [.sup.3 H]
801-receptor association (FIG. 1A) and dissociation (FIG. 2) as
compared to the control level, possibly indicating the presence of
residual glutamate (or glycine) in the membrane preparation. It
seems that AP-5 "freezes" the receptor in such a way that [.sup.3
H] 801 is prevented from penetrating into the channel (see FIG. 1A)
or dissociating from it (FIG. 2). Indeed, the addition of AP-5 to
preequilibrated [.sup.3 H] 801-receptor complexes did not alter the
equilibrium binding (FIG. 2).
The kinetic results together with the equilibrium binding data
indicate that glutamate and glycine affect mainly the rates of
[.sup.3 H] 801-receptor complex formation and dissociation, and not
the maximal number of binding sites (FIG. 1A and FIG. 2) or K.sub.d
(FIG. 2). Thus, in agreement with the electrophysiological data,
[.sup.3 H] 801 like [.sup.3 H]TCP appears to act as a steric
blocker of the NMDA-receptor channel and not as an allosteric
effector.
The findings of this study have important therapeutic implications:
the channel-ligand complexes formed following in vivo
administration of MK-801 can be prevented from dissociating by a
competitive antagonist (e.g. AP-5), which freezes the complex.
FIG. 1A illustrates the time course of association of [.sup.3 H]
801 (5 nM) with the NMDA receptor channel. Binding was determined
at 25.degree. C. as a function of time in the absence () and in the
presence of glutamate and glycine () or in the presence of AP-5 ();
whereas 1B illustrates the observed time constants (K.sub.obs)
derived from the pseudo first order plots of the association of 2,
5 and 8.6 nM [.sup.3 H] 801 in the presence of glutamate and
glycine plotted as a function of the ligand concentration. FIG. 1C
is a first order plot (eq. 2) of [.sup.3 H] 801-receptor
association in the absence of agonists determined with ligand
concentrations of 2 nM () and 8.6 nM ().
FIG. 2 left illustrates the equilibrium binding of [.sup.3 H] 801.
Binding of [.sup.3 H] 801 as a function of its concentration was
assayed after incubation for 4 hr at 25.degree. C. in the absence
() or in the presence () of glutamate and glycine. Also shown are
data for [.sup.3 H] 801 binding to the receptor measured after
their incubation for 3 hr followed by an additional 1 hr of
incubation in the presence of 100 .mu.M AP-5 (). Data are expressed
in the form of Scatchard plot.
FIG. 2 right shows first order plots of the dissociation of [.sup.3
H]MK-801-receptor complexes. Samples were incubated at 25.degree.
C. with 8 nM [.sup.3 H] 801 for 2 hr. The dissociation reaction was
initiated by the addition of 100 .mu.M unlabeled PCP. Data show the
dissociation reaction without added agonists () and in the presence
of 1 .mu.M glutamate (), 1 .mu.M glutamate+1 .mu.M glycine (o), or
100 .mu.M AP-5 (). Reactions were terminated either immediately
(zero time) or at the indicated times. B.sub.o --amount of [.sup.3
H]MK-801 bound at zero time. B.sub.t --amount of [.sup.3 H] 801
bound at time t.
Dosages of the channel blocking agents, such as [.sup.3 H] 801, PCP
and TCP will generally be, when administered by injection, of the
order of 0.2 mg/kg to about 10 mg/kg of patient weight.
The glutamate or similar agent will be administered so as to obtain
open channels; if same is present in adequate excess, such
administration may be unnecessary.
The antagonist to the amino acids, such as AP-5, will be
administered (i.v.) in the order of from about 5 mg/kg to about 100
mg/kg.
In patients suffering from a variety of diseases, the level of the
glutamate will be a high one. Generally this will be such that it
will maintain the channels in an open state. In such cases the
administration of glutamate will not be necessary. In order to
anchor the drug in such channel, the competitive antagonist of the
glutamate or similar amino acid type will be effected with a
certain time delay, of the order of about 5 to 20 minutes after the
drug.
Drugs of the [.sup.3 H] 801, PCP or TCP type will block the
channels after a brief period of time which will be open due to the
presence of the excess of glutamate; and the administration of AP-5
type antagonists will firmly anchor the drug in such channels. The
administration of the AP-5 type compound may be repeated after a
certain period of time in order to bring about a prolonged of the
drug in the channel.
* * * * *